淡江大學機構典藏:Item 987654321/32592
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    题名: 啤酒酵母菌基因ALD2與ALD3之選殖、同源表現、純化，與催化活性之探討
    其它题名: Cloning, homologous expression, purification, and catalytic characterization of ALD2 and ALD3 of saccharomyces cerevisiae
    作者: 李慧玲;Lee, Hui-lin
    贡献者: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    关键词: 醛類脫氫酶;真核細胞;同源表現;親和性層析法;凝血酶;aldehyde dehydorgenase;eukaryotic;homologious expression;affinity chromatography;thrombin
    日期: 2005
    上传时间: 2010-01-11 02:24:49 (UTC+8)
    摘要: 啤酒酵母菌(Saccharomyces cerevisiae)之ALD2 (YMR170C )及ALD3 (YMR169C)，是以二縱排重覆(two tandem-repeated)的ORFs存在其第13號染色體中。這兩個蛋白質，在胺基酸序列的比較上，呈現出87%的同等性及91%的相似性，同時此二蛋白在特徵上被認為是屬於細胞質壓力誘導型(cytoplasmic stress-inducible )-醛類脫氫酶(aldehyde dehydorgenase, ALD)之同功異構酶(isoforms)。根據Navarro-Avino等學者的研究指出，ALD3p可藉由NAD+當輔酶(coenzyme)，並具有以acetaldehyde及betaine aldehyde為受質之酵素活性 (Navarro-Avino et al., 1999)，但是至目前為止，其尚無詳細的酵素動力學被測定出來；此外ALD2p的酵素活性亦尚未被表達與分析之。本研究為了產生具有酵素活性的ALD2p及ALD3p，我們利用真核細胞同源表現系統，將ALD2與ALD3基因構築到具有Glutathione S – Trans ferase (GST)基因的表現載體pEG-KT，利用4 % galactose誘導此二重組蛋白(GST-ALD2、GST-ALD3)在Saccharomyces cerevisiae (BJ2168)中大量表現，並利用GST親合性層析法純化此二蛋白。研究結果得知，我們所建構表現的重組型GST-ALD2、GST-ALD3具有催化propioaldehyde、γ-aminobutyraldehyde、3-aminopropioaldehyde，及betaine aldehyde等不同醛類受質之酵素活性。
    Saccharomyces cerevisiae ALD2 (YMR170C) and ALD3 (YMR169C) are two tandem-repeated ORFs on Chromosome XIII. The amino acid sequence comparison of these two proteins shows significant similarity with 87% identities and 91% positives, and they were characterized as the cytoplasmic stress-inducible isoforms of aldehyde dehydorgenase (ALD). In the previous studies, assays with over-expressed Ald3p showed that this protein is NAD+ linked and active with acetaldehyde and betaine aldehyde. However, none of detailed kinetics has been determined. Neither has ALD2p been expressed and assayed. In this thesis, in order to produce large amount of biologically active Ald2p and Ald3p, the eukaryotic homologous expression system was applied, and the full-length of ALD2 and ALD3 gene was constructed into pEG-KT expression vector. Then the vector was transformed into Saccharomyces cerevisiae (BJ2168), and the transformants were able to over-express recombinant GST-ALD2 and GST-ALD3 by 4% galactose. To study the purified ALD2p and ALD3p, GST affinity chromatography strategy was used. The activity of clarified cell extract was examined by propioaldehyde. And the recombinant protein was characterized by betaine aldehyde, γ-aminobutyraldehyde and 3-aminopropioaldehyde.
    显示于类别:[生命科學研究所] 學位論文

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